In identifying pathways for the targeted therapy of cancer, three requirements are paramount. The pathway must be activated in tumor cells, it must be amenable to pharmacological modulation, and inhibition of the pathway in normal cells must not lead to undue toxicity. STAT family transcription factors are targets that fulfill these requirements fully. In particular, STAT3 and STAT5 are activated inappropriately in a wide array of human tumors, and their inhibition can be tolerated in normal tissue with little detrimental effect. We have developed a system to screen for inhibitors of STAT5, and we have identified a number of promising compounds. However, STAT5, which is activated in many hematologic cancers, is generally not inhibited by the STAT3 modulators we have identified. Given the success we have had in identifying STAT3 inhibitors, and their lack of cross-reactivity with STAT5, the goal of this proposal is to develop a high throughput screening system to identify inhibitors of STAT5. We will create cell lines in which stable transfection is used to introduce a luciferase reporter gene driven by the beta-casein promoter. Luciferase activity is induced from this construct in response to activated STAT5, but not in response to other STATs or other transcription factors. We will use three cellular systems:T47D human breast cancer cells, in which STAT5 can be activated through the endogenous prolactin receptor; NKL human leukemia cells in which STAT5 is activated following IL-2 treatment; and, Ba/F3 cells, a hematopoietic cell line in which STAT5 is activated in response to IL-3. The screen will be conducted at the Institute of Chemistry and Cell Biology at Harvard Medical School, where we have a longstanding collaboration to identify modulators of other STATs. In the initial screen, we will identify compounds that inhibit or induce STAT5-dependent luciferase expression. Although the primary goal of this work is to identify STAT5 inhibitors, compounds that activate STATS may also have therapeutic benefit, particularly in breast cancer. These modulators will then be tested for specificity for the STAT pathway by performing a secondary screen in cells stably expressing NF-kappaB dependent luciferase constructs, which we had constructed previously for our earlier screens. Compounds that show specificity for STATS-dependent gene activation will then be evaluated for mechanistic properties, and will be studied in appropriate in vitro and animal models. Through this validated and structured approach, we plan to identify ten specific inhibitors and five specific activators of STAT5 by the end of the one year funding period. The ultimate goal is the development of compounds that will act as targeted therapies for human cancers. [unreadable] [unreadable]